Reagent useful for synthesizing sulfurized oligonucleotide analogs

ABSTRACT

The present invention is directed to a method of synthesizing sulfurized oligonucleotide analogs by reacting an oligonucleotide analog containing a phosphorous(III) linkage with a dithiocarbonic acid diester polysulfide having the formula ##STR1## to produce a sulfurized oligonucleotide analog. The diester polysulfide reagent is useful in solution and solid phase oligonucleotide analog synthesis.

This application is a Continuation of application Ser. No. 08/811,233,filed on Mar. 3, 1997 now U.S. Pat. No. 5,902,881.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to a method of synthesizing sulfurizedoligonucleotide analogs by reacting an oligonucleotide analog containinga trivalent phosphorous linkage with a dithiocarbonic acid diesterpolysulfide.

2. Discussion of the Background

It is well-known that most of the bodily states in mammals, includingmost disease states, are effected by proteins. By acting directly orthrough their enzymatic functions, proteins contribute in majorproportion to many diseases in animals and man.

Classical therapeutics has generally focused on interactions with suchproteins in an effort to moderate their disease causing or diseasepotentiating functions. Recently, however, attempts have been made todirectly inhibit the production of proteins involved in disease byinteracting with the messenger RNA (mRNA) molecules that direct theirsynthesis. These interactions have involved the hybridization ofcomplementary, or antisense, oligonucleotides or oligonucleotidesanalogs to mRNA. Hybridization is the sequence-specific hydrogen bondingof an oligonucleotide or oligonucleotide analog to an mRNA sequence viaWatson-Crick hydrogen bond formation. Interfering with the production ofproteins involved in disease would provide maximum therapeutic resultswith minimum side effects.

The pharmacological activity of antisense oligonucleotides andoligonucleotide analogs depends on a number of factors that influencethe effective concentration of these agents at specific intracellulartargets. One important factor for oligonucleotides and analogs thereofis their stability to nucleases. It is unlikely that unmodifiedoligonucleotides containing phosphodiester linkages will be usefultherapeutic agents because they are rapidly degraded by nucleases.Modified oligonucleotides which are nuclease resistant are thereforegreatly desired.

Phosphorothioate and phosphorodithioate oligonucleotide analogs whichhave one or both of the non-bridging oxygens of the naturalphosphodiester linkage replaced with sulphur, respectively, areespecially promising antisense therapeutics. These oligonucleotideanalogs are highly resistant to nucleases, have the same charge asnatural phosphodiester-containing oligonucleotides, and are taken up bycells in therapeutically effective amounts. See, for example, Baracchiniet al, U.S. Pat. No. 5,510,239; Ecker, U.S. Pat. No. 5,512,438; Bennettet al, U.S. Pat. No. 5,514,788; and Ecker et al, U.S. Pat. No.5,523,389.

Phosphorothioate and phosphorodithioate oligonucleotide analogs areconveniently synthesized with automated DNA synthesizers using hydrogenphosphonate chemistry which permits the phosphonate backbone to besulfurized in a single step after automated synthesis. One drawback ofthis approach is that coupling yields during chain synthesis aretypically lower than those obtained using phosphoramidite chemistry. Thefinal yield of the desired oligonucleotide analog is therefore too lowdue to the low individual coupling yields.

Automated synthesis using phosphoramidite chemistry is a highlydesirable approach to the synthesis of these sulfurized oligonucleotideanalogs, with coupling yields typically greater than 99%. However, thephosphorous(III)-containing phosphite intermediates are unstable underthe conditions of the detritylation step of the synthesis cycle.Therefore, these phosphorous(III) linkages must be sulfurized after eachcoupling step.

A more recent method for the synthesis of oligonucleotide analogs is the"blockmer" approach. In blockmer synthesis, an oligonucleotide analog ismade by the sequential coupling of short protected oligomers or blocks,e.g., a dinucleotide, on a solid support. This strategy offers severaladvantages over the conventional synthetic approach which involves thesequential coupling of monomeric nucleoside phosphoramidites. The numberof synthesis cycles required to prepare an oligonucleotide analog isreduced, saving time and minimizing reagent consumption. The blocks maybe prepared on a large scale using inexpensive solution phase synthesistechniques. In order to prepare sulfurized oligonucleotide analogs bythe blockmer method, a reagent for sulfuring the phosphorous(III)linkages of the blocks on a large scale is required. The blockmerapproach is described in the following references: Ravikumar et al, WO95/32980; WO 94/15947; Journal of Organic Chemistry 1984, 49, 4905-4912;Helevetica Chimica Acta 1985, 68, 1907-1913; Chem. Pharm. Bull. 1987,35, 833-836.

There are several reagents available for sulfurizing the phosphiteintermediates during automated oligonucleotide synthesis. All of thesereagents have drawbacks which limit their use for synthesizingsulfurized oligonucleotide analogs.

Elemental sulfur, for example, has been used to sulfurizephosphorous(III) linkages in solid phase oligonucleotide synthesis.However, elemental sulphur is not suitable for use with automatedsynthesizers because of its poor solubility in standard solvents andslow sulfurization rate. In addition, carbon disulfide, the preferredsolvent for elemental sulphur, is highly volatile and has a low flashpoint. See, U.S. Pat. Nos. 5,252,723 and 5,449,769.

The Beaucage reagent, 3H-1,2-benzodithiol-3-one, is a considerably moreefficient sulfurizing agent. However, this reagent precipitates fromsolution and clogs the solvent and reagent transfer lines of anautomated DNA synthesizer. Also, the by-product formed during thesulfurization reaction is a potent oxidizing agent. This by-product canlead to side products, e.g., phosphodiesters, which are difficult toseparate from the desired sulfurized oligonucleotides. In addition, thepreparation of this reagent involves expensive and toxic materials, andis therefore not amenable for large-scale synthesis of sulfurizedoligonucleotide analogs. See, U.S. Pat. No. 5,003,097.

Tetraethylthiuram disulfide is an inexpensive and chemically stablesulfurization reagent. However, the sulfurization rate is slow andtherefore a significant molar excess of this reagent is required. Evenwith an excess of this reagent, sulfurization yields are unacceptablylow. See, U.S. Pat. No. 5,166,387.

Phenylacetyl disulfide may be used to sulfurize phosphite intermediatesduring automated oligonucleotide synthesis. However, this reagent hasnot been reported to be useful for large-scale synthesis of sulfurizedoligonucleotide analogs. See, Recherches Travaux Chimiques des Pays-Bas1991, 110, 325-331; Tetrahedron Letters 1989, 30, 6757-6760; Synthesis1981, 637-638.

Accordingly, there remains a need in the art for methods and reagentsfor synthesizing sulfurized oligonucleotide analogs which overcome theseproblems.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method ofsynthesizing a sulfurized oligonucleotide using a reagent thatefficiently sulfurizes a phosphorous(III) linkage in an oligonucleotideanalog.

Another object of the present invention is to provide a method ofsynthesizing a sulfurized oligonucleotide using a reagent that does notrequire the use of solvents having a high volatility and flash point.

Another object of the present invention is to provide a method ofsynthesizing a sulfurized oligonucleotide using a reagent that is highlysoluble in organic solvents and does not precipitate from solution.

Another object of the present invention is to provide a method ofsynthesizing a sulfurized oligonucleotide using a reagent that is usefulin automated solid phase synthesis of oligonucleotide analogs.

Another object of the present invention is to provide a method ofsynthesizing a sulfurized oligonucleotide using a reagent that iscompatible with large-scale and small-scale synthesis using solutionphase methods.

Another object of the present invention is to provide a sulfurizingreagent composition that may be used to sulfurize a phosphorous(III)linkage of an oligonucleotide analog.

These objects and others may be accomplished with a method ofsynthesizing a sulfurized oligonucleotide analog by reacting anoligonucleotide analog containing a phosphorus(III) linkage capable ofbeing sulfurized with a thiodicarbonic acid diester polysulfide havingthe formula: ##STR2## where each R is an inert side chain, and n is 2, 3or 4.

The above objects may also be accomplished with a method of synthesizinga sulfurized oligonucleotide analog by:

(a) providing a nucleoside analog having a blocked hydroxyl group;

(b) deblocking the blocked hydroxyl group to produce a free hydroxylgroup;

(c) reacting the free hydroxyl group with a protected nucleoside analogphosphoramidite or a protected nucleoside analog phosphorothioamiditehaving a blocked hydroxyl group to produce an oligonucleotide analogcontaining a phosphorous(III) linkage and a blocked hydroxyl group;

(d) reacting the phosphorous(III) linkage with a reagent selected fromthe group consisting of an oxidizing agent and a dithiocarbonic aciddiester polysulfide to produce an oxidized or sulfurized phosphorous(V)linkage;

(e) repeating steps (b) through (d) at least once to produce asulfurized oligonucleotide analog, wherein at least one step (d) in themethod is reacting the phosphorous(III) linkage with the dithiocarbonicacid diester polysulfide of the present invention.

The above objects may also be accomplished with a sulfurizing reagentcomposition containing an effective amount of thiodicarbonic aciddiester polysulfide for sulfurizing a phosphorous(III) linkage of anoligonucleotide analog and at least one solvent.

DETAILED DESCRIPTION OF THE INVENTION

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description.

As used in the present invention, the term "oligonucleotide analog"includes linear oligomers of natural or modified nucleosides linked byphosphodiester bonds or analogs thereof ranging in size from twomonomeric units to several hundred monomeric units. Oligonucleotideanalogs include modifications of the heterocyclic base moiety and/or thesugar portion of a component nucleotide. In particular, the termincludes non-natural oligomers containing phosphorus(III) linkages whichare amenable to sulfurization. Preferably, the modifications do notinhibit the ability of an oligonucelotide analog to bind to a targetnucleic acid. The term "sulfurized oligonucleotide analog" is anoligonucleotide analog containing at least one analog of aphosphodiester linkage in which one or both of the non-bridging oxygenatoms are replaced by sulfur. The term "nucleoside analog" refers to anatural or modified nucleoside. In particular, this term includesnucleosides that are modified at the heterocyclic base and/or sugar toenhance hybridization to the target nucleic acids. It is to beunderstood that the stereochemical relationship between the sugarsubstituents in the nucleoside and oligonucleotide analogs disclosedherein is preferably the same as that of naturally-occurring DNA andRNA, see G. M. Blackburn and M. J. Gait (eds.), Nucleic Acids inChemistry and Biology (ILR Press, 1990), Chapter 2, pp. 19-70.

The thiodicarbonic acid diester polysulfide of the present inventionpreferably has the formula: ##STR3## where each R is preferably an inertgroup. These groups preferably do not contain any reactive moietieswhich could lead to side reactions or poor yields in the sulfurizationreaction. Preferably, each R is independently C₁ -C₈ alkyl, substitutedC₁ -C₈ alkyl, C₂ -C₈ heterocycloalkyl containing up to threeheteroatoms, substituted C₂ -C₈ heterocycloalkyl containing up to threeheteroatoms, C₆ -C₁₄ aryl, substituted C₆ -C₁₄ aryl, C₃ -C₁₁ hetarylcontaining up to three heteroatoms, substituted C₃ -C₁₁ hetarylcontaining up to three heteroatoms, C₇ -C₁₈ aralkyl, substituted C₇ -C₁₈aralkyl, C₄ -C₁₅ heterocycloaralkyl containing up to three heteroatomsor substituted C₄ -C₁₅ heterocycloaralkyl containing up to threeheteroatoms. The term "C₁ -C₈ alkyl" includes linear, branched andcyclic alkyl groups. The term "substituted" means that up to threehydrogen atoms in the group are substituted with up to three halogen,nitro, cyano, C₁ -C₈ alkyl, O--C₁ -C₈ alkyl, N--C₁ -C₈ alkyl, S--C₁ -C₈alkyl groups or combinations thereof. More preferably, each R isindependently C₂ -C₈ alkyl, C₆ -C₁₄ aryl or substituted C₆ -C₁₄ aryl.Most preferably, each R is independently ethyl or p-chlorophenyl. Thedithiocarbonic acid diester polysulfide may be a disulfide, trisulfideor tetrasulfide, i.e., n is 2, 3 or 4, respectively. Preferably n is 2or 3, and more preferably, n is 2.

The thiodicarbonic acid diester polysulfide may be prepared by oxidationof the corresponding thiodicarbonic acid or acid salt with oxidizingagents, such as iodine or bromine. Methods of synthesis and propertiesof these polysulfides are described in the following references: Baranyet al, Journal of Organic Chemistry, 1983, 48, 4750-4761; W. F. Zeise,J. Prakt. Chem. 1845, 36, 352-362; Losse et al., J. Prakt. Chem. 1961,13, 260; S. R. Rao, Xanthates and Related Compounds, (M. Decker, NewYork, 1971); Bulmer et al, J. Chem. Soc. 1945, 674-677.

The thiodicarbonic acid diester polysulfide of the present invention isan efficient reagent for sulfurizing phosphorous(III) linkages inoligonucleotide analogs. The reagent does not precipitate out ofsolution, even on prolonged storage. The reagent may be used in solutionphase synthesis and is particularly useful in solid-phase synthesis ofoligonucleotide analogs with automated DNA synthesizers. The reagent isparticularly useful in large-scale synthesis, using either solutionphase or solid phase techniques.

When used to sulfurize phosphorus(III) linkages in oligonucleotideanalogs, the thiodicarbonic acid diester polysulfide is preferablydelivered to the oligonucleotide analog in a suitable organic solvent,such as acetonitrile, pyridine, tetrahydrofuran, dichloromethane,dichloroethane and collidine. These solvents may be used singly or asmixtures in any proportion. Preferable solvents are pyridine,dichloromethane and mixtures thereof. Pyridine is most preferred. Thereagent may be used at any effective concentration for sulfurizing aphosphorous(III) linkage, preferably between 0.01 M to 1.5 M, morepreferably from 0.2 to 1.2 M; and most preferably from 0.5 to 1.0 M.

The sulfurization reaction may be conducted at any convenienttemperature, preferably from 0 to 70° C.; more preferably from 10 to 40°C.; and most preferably at about room temperature, i.e., 18 to 25° C.The sulfurization reaction is preferably conducted for 30 seconds to 15minutes, more preferably, 1 to 15 minutes; and most preferably, 3 to 10minutes. Preferably, sulfurization is performed under anhydrousconditions with the exclusion of air.

The present method for synthesizing a sulfurized oligonucleotide analogmay be applied to any oligonucleotide analog containing at least onephosphorus(III) linkage which is amenable to sulfurization. Inparticular, the present method is useful for sulphurizing phosphitetriesters, thiophosphite triesters, and hydrogen phosphonates. Morepreferably, the phosphorus(III) linkage is a phosphite triester or athiophosphite triester. Most preferably, the phosphorus(III) linkage isa phosphite triester.

Detailed procedures for synthesizing oligonucleotide analogs containingat least one phosphorus(III) linkages are well-known in the art and aredescribed in the following references: M. J. Gait (ed.), OligonucleotideSynthesis, A Practical Approach (ILR Press, 1984); J. S. Cohen (ed.),Oligonucleotides: Antisense Inhibitors of Gene Expression, (CRC Press,Inc., Boca Raton, Fla., 1989).

Preferably, the dithiocarbonic acid diester polysulfide of the presentinvention is used in conjunction with the phosphoramidite orphosphorothioamidite synthetic approaches. Synthesis may be conducted insolution phase or using solid phase techniques. More preferably, thesynthesis is conducted using a solid support. Most preferably, thesynthesis is conducted on a solid support using an automated DNAsynthesizer, e.g., an APPLIED BIOSYSTEMS model 380B or a similarmachine.

Preferably, this synthetic approach involves the following steps: (1)deprotecting a blocked reactive functionality on the growingoligonucleotide analog chain or on the first nucleoside analog monomer,to produce a deblocked reactive functionality, (2) reacting anappropriately blocked and protected nucleoside analog phosphoramidite orphosphorothioamidite monomer with the deblocked reactive functionalityof the growing nucleotide analog chain, preferably in the presence of anactivator, to form an oligonucleotide analog containing aphosphorus(III) linkage, (3) capping any unreacted functionalities, and(4) sulfurizing the newly-formed phosphorus(III) linkage with thethiodicarbonic acid diester polysulfide to obtain the phosphorus atom ina sulfurized pentacoordinate state.

The term "blocked" means that a reactive functionality, usually anucleophile, e.g., a 5' hydroxyl, is protected with a group that may beselectively removed. Preferably, these blocking groups are labile todilute acid, e.g. dichloroacetic acid in dichloromethane, and are stableto base. Preferable blocking groups include 4,4'-dimethoxytrityl (DmTr),monomethoxytrityl, diphenylmethyl, phenylxanthen-9-yl (pixyl) or9-(p-methoxyphenyl)xanthen-9-yl (Mox). The 4,4'-dimethoxytrityl group ismost preferred. Throughout the present disclosure the labile 5' blockinggroup is represented as "R⁶ ".

Any natural or non-natural heterocyclic base may be used in the presentinvention, such as adenine, guanine, cytosine, thymine, uracil,2-aminopurine, inosine, substituted pyrimidines, e.g., 5-methylcytosine,and 5-nitropyrrole. Other suitable heterocyclic bases are described byMerigan et al., U.S. Pat. No. 3,687,808. Preferably, the heterocyclicbase is attached to C-1 of the sugar moiety of nucleoside analogphosphoramidite (1) via a nitrogen of the base. Throughout the presentdisclosure the heterocyclic base is respresented as "B".

During synthesis these heterocyclic groups are preferably protected toprevent any reactive group, e.g., an exocyclic amino group, to preventundesired side reactions. The term "protected" means that reactivemoieties such as exocyclic amino groups, 2'-hydroxyl groups, oxygen orsulfur bonded to phosphorus atoms, and the like, have protective groupswhich are generally removed after synthesis of the oligonucleotideanalog is completed. Preferably, these protective groups are labile to abase and/or a nucleophile. This term also includes oligonucleotide andnucleoside analogs which have groups that do not require suchprotection, e.g., heterocyclic bases such as thymine or abasicnucleosides.

Preferable protecting groups for the heterocyclic bases include baselabile groups. The exocyclic amino groups of the heterocyclic groups arepreferably protected with acyl groups that are removed by base treatmentafter synthesis of the sulfurized oligonucleotide analog. Preferably,these protecting groups are C₂ -C₁₀ acyl groups. N-benzoyl andN-isobutyryl protecting groups are particularly preferred. Adenine ispreferably protected as an N² -isobutyryl derivative. Guanine ispreferably protected as an N⁶ -isobutyryl derivative. Cytidine ispreferably protected as an N⁴ -benzoyl derivative.

The sulfurized nucleotide analogs of the present invention may besubstituted at the 2' position. Preferable 2' substituents are groupsthat enhance the hybridization of an oligonucleotide analog with itstarget nucleic acid, a group that improves the in vivo stability of anoligonucleotide analog or enhances the pharmacokinetic and/orpharamacodynamic properties of an oligonucleotide analog. Examples of 2'substituents include hydrogen, hydroxyl, F, Cl, Br, C₁ -C₈ alkyl,substituted C₁ -C₈ alkyl, C₂ -C₈ heterocycloalkyl containing up to threeheteroatoms, substituted C₂ -C₈ heterocycloalkyl containing up to threeheteroatoms, C₆ -C₁₄ aryl, substituted C₆ -C₁₄ aryl, C₃ -C₁₁ hetarylcontaining up to three heteroatoms, substituted C₃ -C₁₁ hetarylcontaining up to three heteroatoms, C₇ -C₁₈ aralkyl, substituted C₇ -C₁₈aralkyl, C₄ -C₁₅ heterocycloaralkyl containing up to three heteroatoms,substituted C₄ -C₁₅ heterocycloaralkyl containing up to threeheteroatoms, O--C₁ -C₈ alkyl, substituted O--C₁ -C₈ alkyl (such as CF₃),O--C₂ -C₈ heterocycloalkyl containing up to three heteroatoms,substituted O--C₂ -C₈ heterocycloalkyl containing up to threeheteroatoms, O--C₆ -C₁₄ aryl (such as phenyl), substituted O--C₆ -C₁₄aryl, O--C₃ -C₁₁ hetaryl containing up to three heteroatoms, substitutedO--C₃ -C₁₁ hetaryl containing up to three heteroatoms, O--C₇ -C₁₈aralkyl (such as benzyl), substituted O--C₇ -C₁₈ aralkyl, O--C₄ -C₁₅heterocycloaralkyl containing up to three heteroatoms, substituted O--C₄-C₁₅ heterocycloaralkyl containing up to three heteroatoms, O--C₁ -C₈-alkyl-O--C₁ -C₈ -alkyl, O--C₁ -C₈ alkenyl, O--C₁ -C₈ alkoxyamino,O-tri-C₁ -C₈ -alkyl silyl (such as tert-butyldimethylsilyl) substitutedO-tri-C₁ -C₈ -alkyl silyl, NH--C₁ -C₈ alkyl, N--(C₁ --C₈)₂, NH--C₁ -C₈alkenyl, N--(C₁ -C₈)₂ alkenyl, S--C₁ -C₈ alkyl, S--C₁ -C₈ alkenyl, NH₂,N₃, NH--C₁ -C₈ -alkyl-NH₂, polyalkylamino and an RNA cleaving group.Preferable RNA cleaving groups include theO-{3-propoxy-[2-naphthyl-7-(1-(dimethylaminosulfonyl)-imidazol-4-yl)]}group and theO-{3-propoxy-[2-naphthyl-7-(1-(dimethylaminosulfonyl-2-methoxy-5-acetylaminomethyl)-imidazol-4-yl)]}group. These groups are discussed by Cook et al, U.S. Pat. No.5,359,051.

Preferably, the 2' substituents are hydrogen, hydroxyl, O--C₁ -C₈ alkyl,F, O--C₁ -C₈ -alkoxyamino and O--C₁ -C₈ -alkyl-O--C₁ -C₈ -alkyl. Morepreferably, the 2' substituents are hydrogen, O--C₁ -C₈ alkyl, F andO--C₁ -C₈ -alkyl-O--C₁ -C₈ -alkyl. Most preferably, the 2' substituentis hydrogen or a methoxyethoxy group. Throughout the present disclose,the 2' substituent is respresented as "X".

A variety of protecting groups for the oxygen and sulfur atoms attachedto the phosphorus atom in the nucleoside analog phosphoramidite andphosphorothioamidite, respectively, may be used. These protecting groupsare preferably removed at after synthesis is complete. Preferably, theseprotecting groups are labile to a base and/or a nucleophile. Mostprefereably, these protecting groups are removed by aqueous ammoniumhydroxide. Preferable protecting groups are 2-cyanoethyl,4-cyano-2-butenyl, 2-diphenylmethylsilylethyl (DPSE) or a 2-N-amidoethylgroup having the formula R¹ CONR² CHR³ CHR⁴ --. These protecting groupsare preferably removed after synthesis, preferably with an aqueoussolution of ammonia at a temperature between room temperature and 75° C.In the present invention, the terms "phosphodiester linkage","phosphorothioate linkage" and "phosphorodithioate linkage" describethese internucleosidic linkages in protected or unprotected form.Throughout the present disclosure, these phosphorous protecting groupswill be represented as "Pg". An oxygen atom or sulfur atom attached tothe phosphorous atom in a nucleoside analog phosphoramidite orthiophosphoramidite or an oligonucleotide analog is represenated as "Y".Preferably, Y is an oxygen atom.

The 4-cyano-2-butenyl protecting group is removed by δ-elimination,preferably using the standard NH₃ /H₂ O deprotection conditions known inthe art. 4-cyano-2-butenyl-protected nucleoside analog phosphoramiditesmay be prepared with 4-cyano-2-butene-1-ol and appropriately protectednucleoside analogs using known synthetic methodology. The synthesis of4-cyano-2-butene-1-ol is disclosed by Ravikumar et al, SyntheticCommunications 1996, 26(9), 1815-1819.

The 2-diphenylmethylsilylethyl (DPSE) protecting group is described inRavikumar et al., WO 95/04065. This protecting group may be removed bytreatment with a base, preferably aqueous ammonium hydroxide. The DPSEgroup may also be removed with fluoride ion. Preferably, the fluorideion is provided from a salt such as a tetraalkylammonium fluoride, e.g.,tetrabutylammonium fluoride (TBAF) or an inorganic fluoride salt, e.g.,potassium fluoride or cesium fluoride in a solvent such astetrahydrofuran, acetonitrile, dimethoxyethane or water.

The 2-N-amidoethyl group is described in the commonly assignedapplication U.S. patent application Ser. No. 7761-0002-55, U.S. Pat. No.5,760,209 (Title: Protecting Group for Synthesizing oligonucleotideAnalogs, Attorney Docket No. 7761-002-55). The 2-N-amidoethyl group hasthe formula R¹ CONR² CHR³ CHR⁴ -, where R¹ is C₁ -C₈ alkyl, substitutedC₁ -C₈ alkyl, C₂ -C₈ heterocycloalkyl containing up to threeheteroatoms, substituted C₂ -C₈ heterocycloalkyl containing up to threeheteroatoms, C₆ -C₁₄ aryl, substituted C₆ -C₁₄ aryl, C₃ -C₁₁ hetarylcontaining up to three heteroatoms, substituted C₃ -C₁₁ hetarylcontaining up to three heteroatoms, C₇ -C₁₈ aralkyl, substituted C₇ -C₁₈aralkyl, C₄ -C₁₅ heterocycloaralkyl containing up to three heteroatomsor substituted C₄ -C₁₅ heterocycloaralkyl containing up to threeheteroatoms. More preferably, R¹ is C₁ -C₈ alkyl, substituted C₁ -C₈alkyl, C₂ -C₈ heterocycloalkyl containing up to three heteroatoms,substituted C₂ -C₈ heterocycloalkyl containing up to three heteroatoms,C₆ -C₁₄ aryl, substituted C₆ -C₁₄ aryl, C₃ -C₁₁ hetaryl containing up tothree heteroatoms or substituted C₃ -C₁₁ hetaryl containing up to threeheteroatoms. Even more preferably, R¹ is methyl, fluoromethyl,difluoromethyl or trifluoromethyl. Most preferably, R¹ is methyl,trifluoromethyl or phenyl.

The nitrogen atom of the 2-N-amidoethyl group may be unsubstituted,i.e., R² may be hydrogen, or substituted. Preferably, R² is hydrogen, C₁-C₈ alkyl, substituted C₁ -C₈ alkyl, C₂ -C₈ heterocycloalkyl containingup to three heteroatoms, substituted C₂ -C₈ heterocycloalkyl containingup to three heteroatoms, C₆ -C₁₄ aryl, substituted C₆ -C₁₄ aryl, C₃ -C₁₁hetaryl containing up to three heteroatoms, substituted C₃ -C₁₁ hetarylcontaining up to three heteroatoms, C₇ -C₁₈ aralkyl, substituted C₇ -C₁₈aralkyl, C₄ -C₁₅ heterocycloaralkyl containing up to three heteroatomsor substituted C₄ -C₁₅ heterocycloaralkyl containing up to threeheteroatoms. More preferably, R² is hydrogen or C₁ -C₈ alkyl. Mostpreferably, R² is hydrogen or methyl.

The ethyl moiety of the 2-N-amidoethyl group may be unsubstituted, e.g.,R³ and R⁴ may both be hydrogen. Alternatively, the ethyl moiety may besubstituted with groups that preferably do not compromise the stabilityof the 2-N-amidoethyl group during oligonucleotide analog synthesis andpermit the protecting group to be removed by treatment with a baseand/or a nucleophile following step-wise assembly of an oligonucleotideanalog. Preferable R³ and R⁴ groups are hydrogen, C₁ -C₈ alkyl,substituted C₁ -C₈ alkyl, C₂ -C₈ heterocycloalkyl containing up to threeheteroatoms, substituted C₂ -C₈ heterocycloalkyl containing up to threeheteroatoms, C₆ -C₁₄ aryl, substituted C₆ -C₁₄ aryl, C₃ -C₁₁ hetarylcontaining up to three heteroatoms, substituted C₃ -C₁₁ hetarylcontaining up to three heteroatoms, C₇ -C₁₈ aralkyl, substituted C₇ -C₁₈aralkyl, C₄ -C₁₅ heterocycloaralkyl containing up to three heteroatomsor substituted C₄ -C₁₅ heterocycloaralkyl containing up to threeheteroatoms. More preferably, R³ is hydrogen or linear C₁ -C₈ alkyl.Most preferably, R³ is hydrogen or methyl. More preferably, R⁴ ishydrogen, C₆ -C₁₄ aryl, substituted C₆ -C₁₄ aryl, C₃ -C₁₁ hetarylcontaining up to three heteroatoms or substituted C₃ -C₁₁ hetarylcontaining up to three heteroatoms. Most preferably, R⁴ is hydrogen orphenyl. The R³ and R⁴ groups are independently selected, i.e., they maybe the same or different.

Alternatively, R³ and R⁴ ₁ together with the carbon atoms they arebonded to, may form a C₃ -C₈ cycloalkyl group, a substituted C₃ -C₈cycloalkyl group, a C₂ -C₈ heterocycloalkyl group containing up to threeheteroatoms or a substituted C₂ -C₈ heterocycloalkyl group containing upto three heteroatoms. In this embodiment, R³ and R⁴, together with thecarbon atoms they are bonded to, preferably form a C₃ -C₈ cycloalkylgroup or a substituted C₃ -C₈ cycloalkyl group. More preferredcycloalkyl groups are C₄ -C₇ or substituted C₄ -C₇ groups, with C₅ -C₆or substituted C₅ -C₆ groups most preferred. An unsubstituted cycloalkylgroup is particularly preferred. An unsubstituted C₆ cycloalkyl group ismost particularly preferred. The stereochemical relationship between theN-amido group and Y may be cis or trans. A trans relationship ispreferred.

An allyl group is also a preferable protecting group for the oxygen orsulfur atom attached to the phosphorus atom in an oligonucleotideanalog. The allyl protecting group is described in U.S. Pat. No.5,026,838. The term "allyl group" includes allyl, methallyl, crotyl,prenyl, geranyl, cinnamyl and p-chlorocinnamyl groups. The number ofcarbon atoms in these groups is preferably 3 to 10. Preferably, theallyl group is an unsubstituted allyl group. The allyl group may beremoved with a palladium(0) compound and a nucleophilic agent, such asan amine or a formic acid salt, under neutral conditions at roomtemperature. A preferred reagent is tetrakis(triphenylphosphine)palladium(0) and n-butylamine in tetrahydrofuran.

An activator is generally used in the coupling of a deblocked reactivefunctionality on the oligonucleotide or nucleoside analog and thephosphoramidite or phosphorothioamidite monomer. Preferable activatorsare well-known in the art, such as 1H-tetrazole,5-(4-nitrophenyl)-1H-tetrazole and diisopropylamino tetrazolide.1H-Tetrazole is most preferred.

A capping step is preferably used after this coupling reaction topermanently block all uncoupled reactive functionalities. Suitablecapping reagents are well-known in the art. A preferable capping reagentis acetic anhydride/lutidine/THF (1:1:8) with N-methylimidazole/THF.

When synthesis is performed by solution phase methods, the 3' terminalhydroxyl group of an oligonucleotide analog is preferably protected toprevent the 3' hydroxyl group from participating in any undesired sidereactions. Preferably, the terminal 3' hydroxyl group is protected witha group which may be removed selectively without removing any otherprotecting groups. A C₂ -C₁₀ acyl group is preferred. The acetyl orlevulinyl group is more preferred. The levulinyl group is mostpreferred. Throughout the present disclosure this 3' protecting group isrepresented as "R⁷ ".

The sequential addition of nucleoside analog phosphoramidites orphosphorothioamidites may be repeated until an oligonucleotide analoghaving the desired sequence length is obtained. The length of thesulfurized oligonucleotide analog is preferably 2 to 200 monomer units;more preferably, 2 to 100 monomer units; even more preferably, 2 to 50monomer units; and, most preferably, 2 to 25 monomer units. These rangesinclude all subranges therebetween.

In a preferred embodiment of the present invention, a sulfurizedoligonucleotide analog is synthesized on a solid support. Suitable solidsupports include controlled pore glass (CPG); oxalyl-controlled poreglass, see for example Alul et al, Nucleic Acids Research 1991, 19,1527; TENTAGEL Support, see Wright et al, Tetrahedron Letters 1993, 34,3373; POROS, a polystyrene resin available from PERCEPTIVE BIOSYSTEMS;and a polystyrene/ divinylbenzene copolymer. Controlled pore glass isthe most preferred solid support. Throughout the present discloure thesolid support is represented as "S^(p) ".

The oligonucleotide analog is preferably attached to the solid supportby a group which may be easily cleaved to release the oligonucleotideanalog from the solid support when synthesis is complete. Preferably,this group may be cleaved upon exposure to a base and/or a nucleophile.More preferably, the group is an acyl. Most preferably, the group is acarboxyl group esterifed with the terminal 3' hydroxyl group of theoligonucleotide analog. The group linking an oligonucleotide analog to asolid support is represented as "L" in the present disclosure.

The present invention includes sulfurized oligonucleotide analogscontaining phosphorothioate, phosphorodithioate, and phosphodiesterlinkages in any combination. The sulfurized oligonucleotide analogs ofthe present invention may contain only sulfurized linkages, e.g.,phosphorothioate and/or phosphorodithioate. The oligonucleotide analogsmay also contain one or more phosphodiester linkages in addition to thesulfurized linkages. In a preferred embodiment, the sulfurizedoligonucleotide analog contains both phosphorothioate and phosphodiesterlinkages. In another preferred embodiment, the oligonucleotide containsboth phosphorodithioate and phosphodiester linkages.

Phosphodiester linkages are formed by oxidizing a phosphorous(III)linkage with any suitable oxidizing reagent known in the art, e.g., I₂/THF/H₂ O, H₂ O₂ /H₂ O, tert-butyl hydroperoxide or a peracid, such asm-chloroperbenzoic acid. I₂ /THF/H₂ O is a preferred oxidizing agent.

In a preferred embodiment, the oligonucleotide analog containing atleast one phosphorous(III) linkage has the formula: ##STR4## where eachPg is independently a group labile to a base and/or a nucleophile or anallyl group;

R⁶ is a labile blocking group;

each B is independently an unprotected or protected heterocyclic base;

each X is independently selected from the group consisting of hydrogen,hydroxyl, F, Cl, Br, C₁ -C₈ alkyl, substituted C₁ -C₈ alkyl, C₂ -C₈heterocycloalkyl containing up to three heteroatoms, substituted C₂ -C₈heterocycloalkyl containing up to three heteroatoms, C₆ -C₁₄ aryl,substituted C₆ -C₁₄ aryl, C₃ -C₁₁ hetaryl containing up to threeheteroatoms, substituted C₃ -C₁₁ hetaryl containing up to threeheteroatoms, C₇ -C₁₈ aralkyl, substituted C₇ -C₁₈ aralkyl, C₄ -C₁₅heterocycloaralkyl containing up to three heteroatoms, substituted C₄-C₁₅ heterocycloaralkyl containing up to three heteroatoms, O--C₁ -C₈alkyl, substituted O--C₁ -C₈ alkyl, O--C₂ -C₈ heterocycloalkylcontaining up to three heteroatoms, substituted O--C₂ -C₈heterocycloalkyl containing up to three heteroatoms, O--C₆ -C₁₄ aryl,substituted O--C₆ -C₁₄ aryl, O--C₃ -C₁₁ hetaryl containing up to threeheteroatoms, substituted O--C₃ -C₁₁ hetaryl containing up to threeheteroatoms, O--C₇ -C₁₈ aralkyl, substituted O--C₇ -C₁₈ aralkyl, O--C₄-C₁₅ heterocycloaralkyl containing up to three heteroatoms, substitutedO--C₄ -C₁₅ heterocycloaralkyl containing up to three heteroatoms, O--C₁-C₈ -alkyl-O--C₁ -C₈ -alkyl, O--C₁ -C₈ alkenyl, O--C₁ -C₈ alkoxyamino,O-tri-C₁ -C₈ -alkyl silyl, substituted O-tri-C₁ -C₈ -alkenyl, silyl,NH--C₁ -C₈ alkyl, N--(C₁ -C₈)₂, NH--C₁ -C₈ alkenyl, N--(C₁ -C₈)₂alkenyl, S--C_(l) -C₈ alkyl, S--C₁ -C₈ alkenyl, NH₂, N₃, NH--C₁ -C₈-alkyl-NH₂, polyalkylamino and an RNA cleaving group;

each Y is independently O or S;

each Z is independently O or S;

L is a group labile to a nucleophile and/or base;

S^(p) is a solid support; and

m is 0 or a positive integer.

Preferred protecting groups, Pg, include 2-cyanoethyl,4-cyano-2-butenyl, 2-diphenylmethylsilylethyl (DPSE) and a2-N-amidoethyl group.

In a preferred embodiment, the oligonucleotide analog containing aphosphorous(III) linkage contains 2 to 100 monomer units, i.e., m is 0to 98; more preferably, from 2 to 50 monomer units, i.e., m is 0 to 48;and most preferably from 2 to 25 monomer units, i.e., m is 0 to 23.

Treating the above oligonucleotide analog with a dithiocarbonic aciddiester polysulfide affords the corresponding sulfurized oligonucleotideanalog having the formula: ##STR5## where Pg, R⁶, B, X, Y, Z, L, S and mare defined above. Z is an oxygen atom or a sulfur atom. Appropriatechoice of Z allows the internucleosidic linkage containing Z to be aphosphodiester, phosphorothioate or phosphorodithioate linkage,depending on selection of Y. For example, when Y and Z are both oxygenthe linkage is a phosphodiester. When Y is oxygen and Z is sulfur, orvice versa, the linkage is a phosphorothioate. When Y and Z are bothsulfur the linkage is a phosphorodithioate. Preferably, Y is an oxygenatom and Z is a sulfur atom which has been introduced using thedithiocarbonic acid diester polysulfide described above. It is tounderstood that the terms phosphodiester, phosphorothioate andphosphorodithioate refer to the internucleotide linkage after removal ofthe Pg groups.

Following sulfurization, the sequence of the sulfurized oligonucleotideanalog may be further extended. Alternatively, the sulfurizedoligonucleotide analog may be deprotected and removed from the solidsupport to afford the corresponding deprotected sulfurized analog. Thedeprotected sulfurized analog may contain phosphorothioate,phosphorodithioate and phosphodiester linkages, in any combination.

In another preferred embodiment of the present invention, theoligonucleotide analog containing at least one phosphorous(III) linkageis a dinucleotide having the formula: ##STR6## where R⁷ is a grouplabile to a base and/or a nucleophile. Sulfurization of the dinucleotideanalog with the dithiocarbonic acid diester polysulfide affords thecorresponding sulfurized dinucleotide analog having the formula:##STR7##

The present invention also provides a method for synthesizing asulfurized oligonucleotide analog by:

(a) providing a nucleoside analog having a blocked hydroxyl group;

(b) deblocking the blocked hydroxyl group to obtain a free hydroxylgroup;

(c) reacting the free hydroxyl group with a protected nucleoside analogphosphoramidite having a blocked hydroxyl group to produce anoligonucleotide analog containing a phosphorous(III) linkage and ablocked hydroxyl group;

(d) reacting the phosphorous(III) linkage with a reagent selected fromthe group consisting of an oxidizing agent and a dithiocarbonic aciddiester polysulfide having the formula: ##STR8## to produce an oxidizedor sulfurized phosphorous(V) linkage; (e) repeating steps (b) through(d) at least once to produce a sulfurized oligonucleotide analog; wherethe oligonucleotide analog contains at least one sulfurizedphosphorous(V) linkage. Preferably, each phosphorous(V) linkage issulfurized.

A preferred nucleoside analog phosphoramidite having a blocked hydroxylgroup used in step (c) has the formula: ##STR9##

The R⁵ groups are chosen such that the nucleoside analog phosphoramiditepreferably couples efficiently with a reactive group on the on thegrowing oligonucleotide analog chain, e.g., a 5' hydroxyl group, to forma phosphorous(III) internucleotide linkage. The R⁵ groups areindependently selected, i.e., they may be the same or different.Preferable R⁵ groups are C₁ -C₈ alkyl, substituted C₁ -C₈ alkyl, C₂ -C₈heterocycloalkyl containing up to three heteroatoms, substituted C₂ -C₈heterocycloalkyl containing up to three heteroatoms, C₆ -C₁₄ aryl,substituted C₆ -C₁₄ aryl, C₃ -C₁₁ hetaryl containing up to threeheteroatoms, substituted C₃ -C₁₁ hetaryl containing up to threeheteroatoms, C₇ -C₁₈ aralkyl, substituted C₇ -C₁₈ aralkyl, C₄ -C₁₅heterocycloaralkyl containing up to three heteroatoms or substituted C₄-C₁₅ heterocycloaralkyl containing up to three heteroatoms; or both R⁵groups together with the nitrogen atom they are bonded to form a C₂ -C₈heterocycloalkyl group containing up to three heteroatoms, a substituteda C₂ -C₈ heterocycloalkyl group containing up to three heteroatoms, a C₃-C₁₁ hetaryl group containing up to three heteroatoms or a substitutedC₃ -C₁₁ hetaryl group containing up to three heteroatoms. Morepreferably, the R⁵ groups are each a C₁ -C₈ alkyl group or together withthe nitrogen atom they are bonded to form a C₂ -C₈ heterocycloalkylgroup containing up to three heteroatoms. Even more preferably, each R⁵group is a branched C₁ -C₈ alkyl group. Most preferably, both R⁵ groupsare isopropyl.

In a preferred embodiment, the nucleoside analog in step (a) is attachedto a solid support. A preferred nucleoside analog attached to a solidsupport has the formula: ##STR10##

In another embodiment, the oligonucleotide analog containing aphosphorous(III) linkage and a blocked hydroxyl group is attached to asolid support and has the formula: ##STR11## and the resultingsulfurized oligonucleotide analog has the formula: ##STR12##

The sulfurized oligonucleotide analog synthesized on a solid support maybe removed from the support, preferably by base treatment. Preferably,all of the protecting groups are removed during cleavage from the solidsupport. Preferable reagents for cleaving the sulfurized oligonucleotideanalog from the solid support are aqueous ammonium hydroxide andammonia/methanol solutions. The simultaneous deprotection and removal ofthe sulfurized oligonucleotide analog is preferably accomplished inaqueous ammonium hydroxide at a temperature between room temperature,i.e., 18 to 25° C., and 75° C.; more preferably, between roomtemperature and 65° C.; and most preferably, between room temperatureand 60° C. A temperature of 55° C. is particularly preferred. Thedeprotection reaction time is preferably 1 to 30 hours; more preferably,1 to 24 hours; and most preferably, 12-24 hours. The concentration ofammonium hydroxide in the solution used for deprotection is preferably20 to 30% by weight; more preferably, 25 to 30% by weight; and mostpreferably, 28 to 30% by weight.

In a preferred embodiment, the sulfurized oligonucleotide analogreleased from the solid support has the formula: ##STR13## where each Bis preferably an unprotecteted heterocyclic base.

Each internucleotide linkage of the sulfurized oligonucleotide analogreleased from the solid support may be ionized, depending on the pH,temperature and salt conditions. Each internucleotide linkage will beionized in aqueous solution at physiologic pH, temperature and saltconditions, i.e., pH 7.2, 37° C. and about 150 mM monovalent salts.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

EXAMPLES Example 1 Diethyldithiocarbonate Disulfide

Potassium ethyl xanthate (120 g) was dissolved in a minimum amount ofwater. Iodine was added to this solution portionwise at 10° C. until adark brown color persisted. A small amount of aqueous saturated Na₂ S₂O₃ was added to quench the reaction and remove the dark brown color fromthe solution. The resulting solution was extracted with ether. Thecombined ether layers were washed three times with water, dried andconcentrated to afford diethyldithiocarbonate disulfide as a pale yellowsolid.

Example 2 Synthesis of a T--T Phosphorothioate Dimer

100 milligrams (4 mmole) of 5'-0-dimethoxytritylthymidine attached to acontrolled pore glass (CPG) support by an ester linkage was added to aglass reactor, and solution of 2% dichloroacetic acid in dichloromethane(volume/volume) was added to deprotect the 5'-hydroxyl group. Theproduct was first washed with dichloromethane and then withacetonitrile. A 0.2 M solution of5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4 M solution of1H-tetrazole in acetonitrile were added, and allowed to react at roomtemperature for 5 minutes. The product was first washed withacetonitrile, and then a 1 M solution of diethyldithiocarbonatedisulfide in pyridine was added and allowed reacted at room temperaturefor 100 seconds. This sulfurization step was repeated for 100 seconds.The CPG was washed with acetonitrile and then a solution of aceticanhydride/lutidine/THF (1:1:8), and N-methylimidazole/THF was added tocap unreacted 5'-hydroxyl groups. The CPG was then washed withacetonitrile. The CPG was treated with 30% aqueous ammonium hydroxidesolution for 90 minutes. The aqueous solution was filtered, concentratedunder reduced pressure to afford the desired T-T phosphorothioate dimer.

Example 3 Synthesis of a C-T Phosphorothioate Dimer

100 milligrams (4 mmole) of 5'-O-dimethoxytritylthymidine attached to aCPG support by an ester linkage was added to a glass reactor, and asolution of 2% dichloroacetic acid in dichloromethane (volume/volume)was added to deprotect the 5'-hydroxyl group. The CPG was then washedwith acetonitrile. A 0.2 M solution of N⁴-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine-3'-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4 M solution of1H-tetrazole in acetonitrile was added, and allowed to stand at roomtemperature for 5 minutes. The CPG was then washed with acetonitrile,followed by addition of a 1 M solution of diethyldithiocarbonatedisulfide in pyridine. The sulfurization reaction was allowed to procedeat room temperature for 100 seconds. The sulfurization step was repeatedfor an additional 100 seconds. The support was then washed withacetonitrile followed by addition of a solution of aceticanhydride/lutidine/THF (1:1:8), and N-methylimidazole/THF to capunreacted 5'-hydroxyl groups. After capping, the CPG was washed withacetonitrile. The CPG was treated with 30% aqueous ammonium hydroxidesolution for 90 minutes and then incubated at 55° C. for 12 hours. Theaqueous solution was filtered, concentrated under reduced pressure toafford the desired C-T phosphorothioate dimer.

Example 4 Synthesis of G-T Phosphorothioate Dimer

100 milligrams (4 mmole) of 5'-O-dimethoxytritylthymidine attatched to aCPG support by an ester linkage was added to a glass reactor, and a 2%solution of dichloroacetic acid in dichloromethane (volume/volume) wasadded to deprotect the 5'-hydroxyl group. The CPG was washed withdichloromethane and then washed with acetonitrile. A 0.2 M solution ofN²-isobutyrl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine-3'-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4 M solution of1H-tetrazole in acetonitrile was added, and reacted at room temperaturefor 5 minutes. The product was washed with acetonitrile, and then a 1 Msolution of diethyldithiocarbonate disulfide in pyridine was added. Thesulurization reaction was allowed to proceed at room temperature for 100seconds. The sulfurization step was repeated for an additional 100seconds. The support was washed with acetonitrile and then a solution ofacetic anhydride/lutidine/THF (1:1:8), and N-methylimidazole/THF wasadded to cap any unreacted 5'-hydroxyl groups. The CPG was then washedwith acetonitrile. The CPG was treated with 30% aqueous ammoniumhydroxide solution for 90 minutes at room temperature and then incubatedat 55° C. for 1 hour. The aqueous solution was filtered and concentratedunder reduced pressure to give the desired T--T phosphorothioate dimer.

Example 5 Synthesis of a 5'-TTTTTTT-3' Phosphorothioate Heptamer

50 milligrams (2 mmole) of 5'-O-dimethoxytritylthymidine attached to aCPG support by an ester linkage was added to a glass reactor, and a 2%solution of dichloroacetic acid in dichloromethane (volume/volume) wasadded to deprotect the 5'-hydroxyl group. The CPG was then washed withacetonitrile. A 0.2 M solution of5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4 M solution of1H-tetrazole in acetonitrile was added, and allowed to react at roomtemperature for 5 minutes. The CPG was washed with acetonitrile, andthen a 1 M solution of diethyldithiocarbonate disulfide in pyridine wasadded and allowed to react at room temperature for 100 seconds. Thissulfurization step was repeated for 100 seconds. The support was washedwith acetonitrile, and then a solution of acetic anhydride/lutidine/THF(1:1:8), and N-methylimidazole/THF was added to cap any unreacted5'-hydroxyl groups. After capping, the solid support was washed withacetonitrile. This complete cycle was repeated five times to afford theprotected thymidine heptamer. The support containing the compound wastreated with 30% aqueous ammonium hydroxide solution for 90 minutes atroom temperature. The aqueous solution is filtered, and concentratedunder reduced pressure to afford the desired phosphorothioate heptamer5'-TTTTTTT-3'.

Example 6 Synthesis of 5'-d(GACTT)-3' Phosphorothioate Pentamer

50 milligrams (2 mmole) of 5'-O-dimethoxytritylthymidine bound to a CPGcontrolled pore glass support through an ester linkage was added to aglass reactor, and a 2% solution of dichloroacetic acid indichloromethane (volume/volume) was added to deprotect the 5'-hydroxylgroup. The CPG was then washed with acetonitrile. A 0.2 M solution of5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4 M solution of1H-tetrazole in acetonitrile were added, and allowed to react at roomtemperature for 5 minutes. The CPG was washed with acetonitrile, andthen a 1 M solution of diethyldithiocarbonate disulfide in pyridine wasadded and allowed to react at room temperature for 100 seconds. Thissulfurization step was repeated for 100 seconds. The support was washedwith acetonitrile and then a solution of acetic anhydride/lutidine/THF(1:1:8), and N-methylimidazole/THF was added to cap the unreacted5'-hydroxyl groups. After capping, the support was washed withacetonitrile.

A solution of 2% dichloroacetic acid in dichloromethane (volume/volume)was added to deprotect the 5'-hydroxyl group, followed by washing withacetonitrile. A 0.2 M solution of N⁴-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine-3'-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4 M solution of1H-tetrazole in acetonitrile were added, and allowed to react at roomtemperature for 5 minutes. After washing the solid support withacetonitrile, a 1 M solution of diethyldithiocarbonate disulfide inpyridine was added and allowed to react at room temperature for 100seconds. This sulfurization step was repeated for 100 seconds. Thesupport was washed with acetonitrile and then a solution of aceticanhydride/lutidine/THF (1:1:8), and N-methylimidazole/THF was added tocap any unreacted 5'-hydroxyl groups. The support was washed withacetonitrile.

A solution of 2% dichloroacetic acid in dichloromethane (volume/volume)was added to deprotect the 5'-hydroxyl group. The CPG was washed withacetonitrile. A 0.2 M solution of N⁶-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine-3'-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in anhydrous acetonitrile and a 0.4 Msolution of 1H-tetrazole in acetonitrile were added and allowed to reactat room temperature for 5 minutes. The product was washed withacetonitrile, and then a 1 M solution of diethyldithiocarbonatedisulfide in pyridine was added and allowed to react at room temperaturefor 5 minutes. This sulfurization step was repeated for 5 minutes. Thesupport was washed with acetonitrile and then a solution of aceticanhydride/lutidine/THF (1:1:8), and N-methylimidazole/THF was added tocap the unreacted 5'-hydroxyl groups. The product is washed withacetonitrile.

A solution of 2% dichloroacetic acid in dichloromethane (volume/volume)was added to deprotect the 5'-hydroxyl group. The product was washedwith acetonitrile. Then, a 0.2 M solution of N²-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine-3'-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4 M solution of1H-tetrazole in acetonitrile were added, and allowed to react at roomtemperature for 5 minutes. The product was washed with acetonitrile, andthen a 1 M solution of diethyldithiocarbonate disulfide in pyridine wasadded and allowed to react at room temperature for 100 seconds. Thissulfurization step was repeated for 100 seconds. The support was washedwith acetonitrile and then a solution of acetic anhydride/lutidine/THF(1:1:8), and N-methylimidazole/THF was added to cap any unreacted5'-hydroxyl groups. The product was washed with acetonitrile.

The CPG was treated with 30% aqueous ammonium hydroxide solution for 90minutes at room temperature and then incubated at 55° C. for 24 hours.The aqueous solution was filtered, concentrated under reduced pressureto give the desired 5'-d(GACTT)-3' phosphorothioate tetramer.

Example 7 Synthesis of Homo-Thymidine Phosphorothioate 19-mer

A 19-base homo-thymidine phosphorothioate oligonucleotide wassynthesized by the phosphoramidite method on an automated synthesizer(ABI model 390Z, Foster City, Calif.). The standard synthesis protocolwas followed, except that in place of the oxidation step a sulfurizationstep was substituted, and this step preceded the capping step. In otherwords, synthesis consisted of repeated cycles of detritylation,coupling, sulfurization, and capping. Separation of the final productfrom the synthesis column and purification were accomplished usingwell-known methods. The sulfurization step involved exposing the growingchain to a 1 M solution of diethyldithiocarbonate disulfide in pyridinefor 100 seconds at room temperature.

The yield of trityl cation released during the detritylation stepsaveraged 99%. The trityl yield is both a measure of coupling efficiencyand a measure of the extent of sulfurization, since non-sulfurizedtrivalent phosphorus linkages in the oligonucleotide are labile tocleavage during detritylation. The 19-mer was cleaved from the supportand deprotected with concentrated ammonium hydroxide under standardconditions and isolated using techniques well-known in the art.

Example 8 Large-Scale Synthesis of a 20-mer PhosphorothioateOligonucleotide Analog

A 20-base phosphorothioate oligonucleotide analog of sequence5'-TCCCGCCTGTGACATGCATT-3' was synthesized by the phosphormidite methodon an OligoPilot automated DNA synthesizer (available from Pharmacia,Sweden). The standard synthesis protocol was used with the oxidationstep replaced with a sulfurization step with diethyldithiocarbonatedisulfide. Sulfurization of each phosphorous(III) linkage wasaccomplished by exposing the oligonucleotide analog to a 0.2 M solutionof diethyldithiocarbonate disulfide in pyridine/dichloromethane (1:1v/v) for 100 seconds at room temperature. The resulting 20-merphosphorothioate oligonucleotide analog was deprotected and cleavagefrom the solid support with aqueous ammonium hydroxide and purified bywell-known methods.

All references cited in the present application are hereby incorporatedby reference in their entirety.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. A method of synthesizing a sulfurizedoligonucleotide analog, comprising:contacting an oligonucleotide analogcomprising at least one phosphorous(III) linkage with a solutioncomprising a solvent and a thiodicarbonic acid diester polysulfiderepresented by the formula: ##STR14## in an automated DNA synthesizer,wherein the solution is in fluid communication with the oligonucleouideanalog on the DNA synthesizer, to produce an oligonucleotide analogcomprising at least one sulfurized phosphorous(V) linkage, wherein eachR is independently selected from the group consisting of C₁ -C₈ alkyl,substituted C₁ -C₈ alkyl, C₂ -C₈ heterocycloalkyl containing up to threeheteroatoms, substituted C₂ -C₈ heterocycloalkyl containing up to threeheteroatoms, C₆ -C₁₄ aryl, substituted C₆ -C₁₄ aryl, C₃ -C₁₁ hetarylcontaining up to three heteroatoms, substituted C₃ -C₁₁ hetarylcontaining up to three heteroatoms, C₇ -C₁₈ aralkyl, substituted C₇ -C₁₈aralkyl, C₄ -C₁₅ heterocycloaralkyl containing up to three heteroatomsand substituted C₄ -C₁₅ heterocycloaralkyl containing up to threeheteroatoms; and n is 2, 3 or
 4. 2. The method of claim 1, wherein eachR is ethyl.
 3. The method of claim 1, wherein said phosphorus(III)linkage is a phosphite triester or a thiophosphite triester.
 4. Themethod of claim 1, wherein each R is independently selected from thegroup consisting of C₂ -C₈ alkyl, substituted C₂ -C₈ alkyl, C₆ -C₁₄ aryland substituted C₆ -C₁₄ aryl; andn is
 2. 5. The method of claim 1,wherein the solvent is selected from the group consisting ofacetonitrile, tetrahydrofuran, collidine, dichloromethane,dichloroethane, pyridine, and mixtures thereof.
 6. The method of claim5, wherein the concentration of the thiodicarbonic acid diesterpolysulfide in the solvent is 0.01 to 1.5 M.
 7. The method of claim 5,wherein the concentration of the thiodicarbonic acid diester polysulfidein the solvent is 0.2 to 1.2 M.
 8. The method of claim 1, wherein theoligonucleotide analog is attached to a solid support.
 9. The method ofclaim 8, wherein the oligonucleotide analog comprising aphosphorous(III) linkage and attached to a solid support is representedby the formula: ##STR15## wherein each Pg is independently a grouplabile to a base and/or a nucleophile or an allyl group;R⁶ is a labileblocking group; each B is independently an unprotected or protectedheterocyclic base; each X is independently selected from the groupconsisting of hydrogen, hydroxyl, F, Cl, Br, C₁ -C₈ alkyl, substitutedC₁ -C₈ alkyl, C₂ -C₈ heterocycloalkyl containing up to threeheteroatoms, substituted C₂ -C₈ heterocycloalkyl containing up to threeheteroatoms, C₆ -C₁₄ aryl, substituted C₆ -C₁₄ aryl, C₃ -C₁₁ hetarylcontaining up to three heteroatoms, substituted C₃ -C₁₁ hetarylcontaining up to three heteroatoms, C₇ -C₁₈ aralkyl, substituted C₇ -C₁₈aralkyl, C₄ -C₁₅ heterocycloaralkyl containing up to three heteroatoms,substituted C₄ -C₁₅ heterocycloaralkyl containing up to threeheteroatoms, O--C₁ -C₈ alkyl, substituted O--C₁ -C₈ alkyl, O--C₂ -C₈heterocycloalkyl containing up to three heteroatoms, substituted O--C₂-C₈ heterocycloalkyl containing up to three heteroatoms, O--C₆ -C₁₄aryl, substituted O--C₆ -C₁₄ aryl, O--C₃ -C₁₁ hetaryl containing up tothree heteroatoms, substituted O--C₃ -C₁₁ hetaryl containing up to threeheteroatoms, O--C₇ -C₁₈ aralkyl, substituted O--C₇ -C₁₈ aralkyl, O--C₄-C₁₅ heterocycloaralkyl containing up to three heteroatoms, substitutedO--C₄ -C₁₅ heterocycloaralkyl containing up to three heteroatoms, O--C₁-C₈ -alkyl-O--C₁ -C₈ -alkyl, O--C₁ -C₈ alkenyl, O--C₁ -C₈ alkoxyamino,O-tri-C₁ -C₈ -alkyl silyl, substituted O-tri-C₁ -C₈ -alkyl silyl, NH--C₁-C₈ alkyl, N--(C₁ -C₈)₂, NH--C₁ -C₈ alkenyl, N--(C₁ -C₈)₂ alkenyl, S--C₁-C₈ alkyl, S--C₁ -C₈ alkenyl, NH₂, N₃, NH--C₁ -C₈ -alkyl-NH₂,polyalkylamino and an RNA cleaving group; each Y is independently O orS; each Z is independently O or S; L is a group labile to a nucleophileand/or base; S^(p) is a solid support; and m is 0 or a positive integer.10. The method of claim 9, whereineach R is independently selected fromthe group consisting of C₂ -C₈ alkyl, substituted C₂ -C₈ alkyl, C₆ -C₁₄aryl and substituted C₆ -C₁₄ aryl; n is 2; and m is an integer between 0and
 198. 11. The method of claim 9, wherein X is hydrogen.
 12. Themethod of claim 9, whereineach Pg is independently selected from thegroup consisting of β-cyanoethyl, 4-cyano-2-butenyl,2-diphenylmethylsilyl and a 2-N-amidoethyl group having the formula R¹CONR² CHR³ CHR⁴ --; each R¹ is independently selected from the groupconsisting of C₁ -C₈ alkyl, substituted C₁ -C₈ alkyl, C₂ -C₈heterocycloalkyl containing up to three heteroatoms, substituted C₂ -C₈heterocycloalkyl containing up to three heteroatoms, C₆ -C₁₄ aryl,substituted C₆ -C₁₄ aryl, C₃ -C₁₁ hetaryl containing up to threeheteroatoms, substituted C₃ -C₁₁ hetaryl containing up to threeheteroatoms, C₇ -C₁₈ aralkyl, substituted C₇ -C₁₈ aralkyl, C₄ -C₁₅heterocycloaralkyl containing up to three heteroatoms and substituted C₄-C₁₅ heterocycloaralkyl containing up to three heteroatoms; each R², R³and R⁴ is independently selected from the group consisting of hydrogen,C₁ -C₈ alkyl, substituted C₁ -C₈ alkyl, C₂ -C₈ heterocycloalkylcontaining up to three heteroatoms, substituted C₂ -C₈ heterocycloalkylcontaining up to three heteroatoms, C₆ -C₁₄ aryl, substituted C₆ -C₁₄aryl, C₃ -C₁₁ hetaryl containing up to three heteroatoms, substituted C₃-C₁₁ hetaryl containing up to three heteroatoms, C₇ -C₁₈ aralkyl,substituted C₇ -C₁₈ aralkyl, C₄ -C₁₅ heterocycloaralkyl containing up tothree heteroatoms and substituted C₄ -C₁₅ heterocycloaralkyl containingup to three heteroatoms, or R³ and R⁴ together with the carbon atomsthey are bonded to form a C₃ -C₈ cycloalkyl group, a substituted C₃ -C₈cycloalkyl group, a C₂ -C₈ heterocycloalkyl group containing up to threeheteroatoms or a substituted C₂ -C₈ heterocycloalkyl group containing upto three heteroatoms; R⁶ is selected from the group consisting of4,4'-dimethoxytrityl, monomethoxytrityl, diphenylmethyl,phenylxanthen-9-yl and 9-(p-methoxyphenyl)xanthen-9-yl; and each B isindependently selected from the group consisting of adenine, guanine,cytosine, thymine, uracil, 2-aminopurine, inosine and 5-methylcytosine,where the exocyclic amino group of each base is protected with an acylgroup.
 13. A composition for synthesizing sulfurized oligonucleotideanalogs, comprising:a thiodicarbonic acid diester polysulfiderepresented by the formula: ##STR16## in contact with a solid supportsuitable for synthesizing oligonucleotide analogs to which is attachedthereto an oligonucleotide analog comprising a phosphorous(III) linkagerepresented by the formula: ##STR17## wherein each R is independentlyselected from the group consisting of C₁ -C₈ alkyl, substituted C₁ -C₈alkyl, C₂ -C₈ heterocycloalkyl containing up to three heteroatoms,substituted C₂ -C₈ heterocycloalkyl containing up to three heteroatoms,C₆ -C₁₄ aryl, substituted C₆ -C₁₄ aryl, C₃ -C₁₁ hetaryl containing up tothree heteroatoms, substituted C₃ -C₁₁ hetaryl containing up to threeheteroatoms, C₇ -C₁₈ aralkyl, substituted C₇ -C₁₈ aralkyl, C₄ -C₁₅heterocycloaralkyl containing up to three hetero atoms and substitutedC₄ -C₁₅ heterocycloaralkyl containing up to three heteroatoms;n is 2, 3or 4 each Pg is independently a group labile to a base and/or anucleophile or an allyl group; R⁶ is a labile blocking group; each B isindependently an unprotected or protected heterocyclic base; each X isindependently selected from the group consisting of hydrogen, hydroxyl,F, Cl, Br, C₁ -C₈ alkyl, substituted C₁ -C₈ alkyl, C₂ -C₈heterocycloalkyl containing up to three heteroatoms, substituted C₂ -C₈heterocycloalkyl containing up to three heteroatoms, C₆ -C₁₄ aryl,substituted C₆ -C₁₄ aryl, C₃ -C₁₁ hetaryl containing up to threeheteroatoms, substituted C₃ -C₁₁ hetaryl containing up to threeheteroatoms, C₇ -C₁₈ aralkyl, substituted C₇ -C₁₈ aralkyl, C₄ -C₁₅heterocycloaralkyl containing up to three heteroatoms, substituted C₄-C₁₅ heterocycloaralkyl containing up to three heteroatoms, O--C₁ -C₈alkyl, substituted O--C₁ -C₈ alkyl, O--C₂ -C₈ heterocycloalkylcontaining up to three heteroatoms, substituted O--C₂ -C₈heterocycloalkyl containing up to three heteroatoms, O--C₆ -C₁₄ aryl,substituted O--C₆ -C₁₄ aryl, O--C₃ -C₁₁ hetaryl containing up to threeheteroatoms, substituted O--C₃ -C₁₁ hetaryl containing up to threeheteroatoms, O--C₇ -C₁₈ aralkyl, substituted O--C₇ -C₁₈ aralkyl, O--C₄-C₁₅ heterocycloaralkyl containing up to three heteroatoms, substitutedO--C₄ -C₁₅ heterocycloaralkyl containing up to three heteroatoms, O--C₁-C₈ -alkyl-O--C₁ -C₈ -alkyl, O--C₁ -C₈ alkenyl, O--C₁ -C₈ alkoxyamino,O-tri-C₁ -C₈ -alkyl silyl, substituted O-tri-C₁ -C₈ -alkyl silyl, NH--C₁-C₈ alkyl, N--(C₁ -C₈)₂, NH--C₁ -C₈ alkenyl, N--(C₁ -C₈)₂ alkenyl, S--C₁-C₈ alkyl, S--C₁ -C₈ alkenyl, NH₂, N₃, NH--C₁ -C₈ -alkyl-NH₂,polyalkylamino and an RNA cleaving group; each Y is independently O orS; each Z is independently O or S; L is a group labile to a nucleophileand/or base; S^(p) is a solid support; and m is 0 or a positive integer.14. The composition of claim 13, wherein each R is independentlyselected from the group consisting of C₂ -C₈ alkyl, substituted C₂ -C₈alkyl, C₆ -C₁₄ aryl and substituted C₆ -C₁₄ aryl; andn is
 2. 15. Thecomposition of claim 13, wherein the solid support is controlled poreglass, a polystyrene resin or a polystyrene/divinylbenzene copolymerresin.
 16. The composition of claim 13, whereineach R is independentlyselected from the group consisting of C₂ -C₈ alkyl, substituted C₂ -C₈alkyl, C₆ -C₁₄ aryl and substituted C₆ -C₁₄ aryl; n is 2; and m is aninteger between 0 and
 198. 17. The composition of claim 13, wherein X ishydrogen.
 18. The composition of claim 13, whereineach Pg isindependently selected from the group consisting of β-cyanoethyl,4-cyano-2-butenyl, 2-diphenylmethylsilyl and a 2-N-amidoethyl grouphaving the formula R¹ CONR² CHR³ CHR⁴ --; each R¹ is independentlyselected from the group consisting of C₁ -C₈ alkyl, substituted C₁ -C₈alkyl, C₂ -C₈ heterocycloalkyl containing up to three heteroatoms,substituted C₂ -C₈ heterocycloalkyl containing up to three heteroatoms,C₆ -C₁₄ aryl, substituted C₆ -C₁₄ aryl, C₃ -C₁₁ hetaryl containing up tothree heteroatoms, substituted C₃ -C₁₁ hetaryl containing up to threeheteroatoms, C₇ -C₁₈ aralkyl, substituted C₇ -C₁₈ aralkyl, C₄ -C₁₅heterocycloaralkyl containing up to three heteroatoms and substituted C₄-C₁₅ heterocycloaralkyl containing up to three heteroatoms, each R², R³and R⁴ is independently selected from the group consisting of hydrogen,C₁ -C₈ alkyl, substituted C₁ -C₈ alkyl, C₂ -C₈ heterocycloalkylcontaining up to three heteroatoms, substituted C₂ -C₈ heterocycloalkylcontaining up to three heteroatoms, C₆ -C₁₄ aryl, substituted C₆ -C₁₄aryl, C₃ -C₁₁ hetaryl containing up to three heteroatoms, substituted C₃-C₁₁ hetaryl containing up to three heteroatoms, C₇ -C₁₈ aralkyl,substituted C₇ -C₁₈ aralkyl, C₄ -C₁₅ heterocycloaralkyl containing up tothree heteroatoms and substituted C₄ -C₁₅ heterocycloaralkyl containingup to three heteroatoms, or R³ and R⁴ together with the carbon atomsthey are bonded to form a C₃ -C₈ cycloalkyl group, a substituted C₃ -C₈cycloalkyl group, a C₂ -C₈ heterocycloalkyl group containing up to threeheteroatoms or a substituted C₂ -C₈ heterocycloalkyl group containing upto three heteroatoms; R⁶ is selected from the group consisting of4,4'-dimethoxytrityl, monomethoxytrityl, diphenylmethyl,phenylxanthen-9-yl and 9-(p-methoxyphenyl)xanthen-9-yl; and each B isindependently selected from the group consisting of adenine, guanine,cytosine, thymine, uracil, 2-aminopurine, inosine and 5-methylcytosine,where the exocyclic amino group of each base is protected with an acylgroup.